After a myocardial infarction, the subsequent cardiomyocyte necrosis stimulates a wound healing response characterized by migration of inflammatory cells into the affected myocardium, extracellular matrix degradation, and neovascularization. Each of these components appears to require activation of latent metalloproteinases by plasmin, which is derived from plasminogen through activation by urokinase-type plasminogen activator (u-PA) expressed on the surface of the infiltrating bone-marrow derived cells (1-3). More recently, it has been suggested that the role of cell surface u-PA may be to facilitate exposure of cryptic cell attachment sites necessary for cell migration (4). This appears to involve direct interaction between u-PA and plasminogen activator inhibitor-1 (PAI-1), which, in its native state, is complexed to vitronectin (4,5). Reaction of PAI-1 with a proteinase, such as u-PA, results in a rapid conformational change that causes it to dissociate from vitronectin and increase its affinity for the low density lipoprotein receptor (6), leading to its clearance and degradation. Removal of PAI-1 from vitronectin exposes the epitope on vitronectin necessary for binding to another of its ligands, the integrin alpha v beta 3 (4,7). Since interactions between the cell surface integrin alpha v beta 3 and tissue vitronectin have been shown to be important in the development of angiogenesis (4,8,9), we hypothesized that excessive PAI-1 protein expression after myocardial infarction might prevent optimal neovascularization by bone marrow-derived angioblasts and that inhibition of PAI-1 expression would promote neovascularization.
In order to develop an approach to inhibit PAI-1 expression which would have clinical applicability, we examined various potential strategies for inhibiting specific mRNA activity.
Antisense oligonucleotides hybridize with their complementary target site in mRNA, blocking translation to protein by sterically inhibiting ribosome movement or by triggering cleavage by endogenous RNAse H (10). Although current constructs are made more resistant to degradation by serum through phosphorothioate linkages, non-specific biological effects due to “irrelevant cleavage” of non-targeted mRNA remains a major concern (11).
Ribozymes are naturally-occurring RNA molecules that contain catalytic sites, making them more potent agents than antisense oligonucleotides. However, wider use of ribozymes has been hampered by their susceptibility to chemical and enzymatic degradation and restricted target site specificity (12).
A new generation of catalytic nucleic acids has been described containing DNA molecules with catalytic activity for specific RNA sequences (13-16). These DNA enzymes exhibit greater catalytic efficiency than hammerhead ribozymes, producing a rate enhancement of approximately 10 million-fold over the spontaneous rate of RNA cleavage, offer greater substrate specificity, are more resistant to chemical and enzymatic degradation, and are far cheaper to synthesize.